Project Summary/Abstract Cholangiocytes are the target cells in cholangiopathies; such as, primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC), which are characterized by the damage and proliferation of cholangiocytes. In cholestatic liver diseases, cholangiocytes, through the products of their cellular activation, are implicated as the key link between bile duct injury and the subepithelial fibrosis that characterizes chronic hepatobiliary injury. While advances have been made to further our understanding of the neuroendocrine factors that modulate biliary proliferation and fibrosis during cholestasis, unfortunately, viable therapies for the management of cholangiopathies remain elusive. Our previous studies have revealed that cholangiocytes express the enzymes necessary to synthesize and secrete melatonin and that melatonin plays a role in regulating biliary hyperplasia during cholestasis. Our overall objective is to determine the molecular mechanisms by which biliary proliferation and fibrosis are regulated by the melatonin/MT1/MT2/GPR50 signaling axis. Our central hypothesis, based upon the strong preliminary data, is that biliary proliferation and fibrosis are differentially regulated by the melatonin/melatonin receptor (MT1 or MT2)-signaling axis in the presence/absence of MT1 or MT2. The central hypothesis was formulated on the basis that knockout of MT1 and MT2 resulted in differential responses of the biliary epithelium to extrahepatic cholestasis. As such, we found: (i) a significant increase of biliary proliferation and fibrosis in MT2 knockout mice during BDL; and (ii) a substantial reduction in biliary proliferation and fibrosis in MT1 knockout mice following BDL. miRNA PCR array analysis of cholangiocytes from BDL MT2 knockout and Mdr2-/- mice exposed to prolonged darkness (that increases melatonin secretion) revealed alterations in the expression levels of miRNAs (miR- 125b, let-7a, miR-200b and miR-181a/b) that will be evaluated as potential targets to regulate biliary proliferation and fibrosis through the melatonin/MT1/MT2/GPR50 signaling axis. To test our central hypothesis, we propose two Specific Aims: (1) disruption of the melatonin/MT1/MT2/GPR50 signaling axis alters biliary proliferation and liver fibrosis during cholestasis and (2) biliary proliferation and liver fibrosis is regulated by the melatonin/MT1/MT2/GPR50 signaling axis via miRNA-regulated proliferative and pro- fibrogenic mechanisms. The successful completion of the studies may provide tools for the development of novel treatment paradigms targeting the melatonin/MT1/MT2/GPR50 signaling axis during chronic liver diseases.